Eukaryotic chromosomal replication is an intricate process that requires the coordinated and tightly regulated action of numerous molecular machines. Failure to ensure once only replication initiation per cell cycle can result in uncontrolled proliferation and genomic instability, two hallmarks of tumor genesis. The origin recognition complex (ORC), first discovered in yeast, is a six-subunit protein machine conserved in all eukaryotes. Yeast ORC constitutively binds to and marks the replication origin throughout the cell cycle. Licensing of the DNA replication origin starts when the critical cell division cycle protein Cdc6p binds to ORC. Recent biochemical studies with purified components indicate that Cdc6p and specific origin DNA sequence activate an ATPase switch in ORC. This induces an extended pre-replication complex (pre-RC)-like nuclease protection footprint on origin DNA that was previously observed only in vivo. Our preliminary EM work reveals a ring-like structural feature in the ORC-Cdc6p complex that is similar in size to the presumptive replicative hexameric MCM helicase. This result supports the emerging concept that the helicase is loaded by replication initiators in a mechanism similar to the loading of the DNA polymerase clamp PCNA by the RF-C clamp loader complex. The formation of the extended pre-RC-like footprint by ORC and Cdc6p, a crucial event in replication origin licensing, is ATP-binding and -hydrolysis dependent. We are interested in revealing the structural basis of this ATPase switch in ORC by studying the structures of ORC-Cdc6p-DNA in the ATP-bound form where the pre-RC footprint is formed, and in a non-hydrolyzable ATP (ATPgammaS)-bound form where the extended footprint is not formed. We hypothesize that the extended pre-RC footprint is a result of Cdc6p-induced origin DNA bending around ORC. We plan to test this hypothesis by determining the approximate binding sites of the three critical elements (A, B1 and B2) of the yeast ARS1 origin DNA. The origin DNA fragments will be biotinylated and labeled with streptavidin before binding with ORC and Cdc6p for EM analysis. These detailed structural studies will provide a long-awaited molecular mechanism for the origin licensing stage of the intricate replication initiation process. [unreadable] [unreadable] [unreadable]